ROS and UV Induced Mitochondria DNA Mutation and Common Deletion In Skin

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Mitochondria are cellular organelles that produce energy (ATP) by consuming oxygen. In the chronological aging process, mitochondria not only produce less ATP, but they also increase the production of reactive oxygen species (ROS) as by-products of aerobic metabolism, particularly during the last step of ADP conversion to ATP. Age-related respiratory function decline can result in enhanced production of ROS in mitochondria. Moreover, the activities of free radical-scavenging enzymes are altered in the aging process. The concurrent age-related changes of these two systems result in the accumulation of endogenous source of ROS in aging tissues. UV irradiation induce an immediate production and accumulation of excess ROS in the skin. The exposure of skin to UV is known to stimulate the intracellular production of ROS (e.g. superoxide and hydrogen peroxide) and reactive nitrogen species (RNS; e.g. nitric oxide). In vitro, ex vivo studies have found that ROS such as oxygen radical, hydrogen peroxide (H2O2), superoxide anion and nitric oxide radical are generated after absorption of UV radiation by chromophores that are also found in keratinocytes (urocanic acid, riboflavin, reduced form of nicotinamide adenine dinucleotide–reduced nicotinamide adenine dinucleotide phosphate [NADH/NADPH], tryptophan). ROS are known to damage proteins, membranes and DNA by oxidation. Continuous generation and accumulation of endogenous and UV induced ROS damages mitochondrial DNA (mtDNA) – deletions, (point) mutations, tandem duplications of mtDNA, recombination. Although UVA penetrates skin more deeply, UVB is more mutagenic. UVA induced DNA mutation is mainly mediated through ROS oxidation. UVB can be absorbed by DNA bases and can induce DNA mutation without ROS; the most common UVB-induced DNA modification is the formation of dimeric photoproducts between adjacent pyrimidines on the same strand of DNA.

The accumulation of mitochondrial DNA (mtDNA) mutations and deletion in intrinsic and extrinsic skin aging is one of the main causes of aging and skin aging. mtDNA is a circular double-stranded DNA 16 559 bp in length. Typically, there are 100–1000 mitochondria per cell and each mitochondrion carries 1–10 copies of the mtDNA. Part of the cellular respiratory electron transport chain membrane bound protein and protein complexes genes are encoded by mitochondria DNA instead of nuclear DNA genome. mtDNA mutation and deletion disrupts the cellular energy production function of the mitochondria. Continuous generation and accumulation of endogenous and UV induced ROS damages mitochondrial DNA (mtDNA) more severely than that of genomic DNA primarily because mitochondria is the cellular organelle where respiratory electron transport chain is located and where free radicals are generated. Mitochondria do not contain any repair mechanism for DNA mutations and deletion except the base excision mechanism; and mtDNA has a lack of association with protective histones protein, making mtDNA more vulnerable to ROS than that of nuclear genome. In fact, the mutation frequency of mtDNA is approximately 50-times of nuclear DNA. Because mtDNA does not contain introns, with coding sequences (genes) being continuous or having very few bases between them; 95% of mtDNA is encoding (functional genes) in comparison to 3% of nuclear DNA, and so any mutagenesis to mtDNA will disrupt a coding gene sequence. The only non-coding region is the ∼1-kb D-loop (displacement loop) which functions as the major regulatory region for both replication and transcription of the genome.

Mutations of mtDNA and the decline of mitochondrial functions, particularly the energy production, is one of the significant causative factor in the chronological, photo aging and age-related disease process. In contrast to nuclear genes, mtDNA is present in multiple copies,each coding genes may exist thousands of copies per cell. Mutant mtDNA and wild-type mtDNA coexist in a cell, a a phenomenon known as heteroplasmy. mtDNA mutations are functionally recessive, cellular dysfunction only occurs when the ratio of mutated to wild-type mtDNA exceeds certain threshold level. A mitochondrial genome harboring a sequence mutation may be replicated allowing its level in cells to increase by intracellular drift. In this way, mtDNA mutations can accumulate during aging

Photoaged skin is characterized by increased mutations of the mitochondrial genome. Of the spectrum of mtDNA deletions identified in the sun-exposed skin, the major deletion have been the 4977 bp common deletion and a 3895 bp deletion. These mtDNA deletions can be also be induced in human skin and cultured skin cells by sub-lethal repetitive doses of UV. It has been shown that a 4977-bp portion of mtDNA containing coding sequences of elements that participate in the respiratory chain is consistently deleted in many different tissues with increasing age and in many age-related diseases. Studies have revealed that the so-called common deletion, a 4,977 base pair deletion of mtDNA, is increased about ten times in photoaged skin compared to that of sun-protected skin of the same individual. Apart from deletions, more tandem mtDNA duplications has been observed in sun-exposed human skin. Large amount of accumulation of mtDNA point mutations in the non-coding regulatory region was also observed in skin. The accumulation of a most common regulatory region point mutation (T414G) in aging skin is shown to be accelerated by UV exposure in human skin.

It was shown that UV-induced mutations of mtDNA in cultured dermal fibroblasts correlates with the amount of ROS production. ROS generated damage of respiratory genes, causing a defective cellular respiratory chain, in a vicious cycle, inducing even more ROS and subsequently allowing mtDNA mutation without the inducing agents which further accelerate the rate of mtDNA damage. In vivo evidence revealed the magnified increase of mtDNA common deletion after the UV irradiation.

Induction of the common deletion in human skin fibroblasts is paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase MMP-1, while TIMP remains, suggesting a causative role of mtDNA mutagenesis in photo skin aging. These observations suggest a link not only between mutations of mtDNA and cellular energy metabolism, but also between mtDNA mutagenesis, energy metabolism, and nuclear encoded gene regulation/activation of the skin fibroblast such as increased skin matrix degradation enzyme MMPs. A number of genes may be activated in response to mtDNA damage signal i.e. DNA damage response genes, although ROS also acts as a signal to regulate and activate genes responsible for the aging signs of the skin. Studies indicate that the mtDNA common deletions in human dermal fibroblasts is causally related to photo skin aging phenotypes because it leads to an altered gene profile in these cells and subsequently to structural and functional alterations of the human dermis which are characteristic of photo skin aging.

NF-kB and AP-1 Molecular Signaling Mechanisms of Intrinsic And Extrinsic Skin Aging

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The molecular signaling pathways associated with skin aging is most and initially studied in the photo skin aging. Numerous researches published combined the pieces of discovery into an general overview picture about what occurred molecularly in photo skin aging. It is generally believed the molecular and cellular changes associated with chronological skin aging is basically parallel those seen in photoaging, but are generally less severe. In addition, other environmental stressors may initiate molecular pathways very similar to those discovered in sun-exposed skin. Multiple cascades of signal transduction pathways involved in the intrinsic and extrinsic aging generally converge into the regulation and activation of two transcription factors – NF-kB and AP-1. NF-kB regulate and activate genes in immune response and is involved in what is know as inflammaging while AP-1 regulate and activate genes that result in the increased extracellular matrix fiber network degradation (collagen breakdown) and decreases the production of new collagen.

UV irradiation of skin in vivo results first in increased levels of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) in skin. Excesses of ROS, which result from aerobic metabolism, accumulate over time during chronological aging as well. H2O2 can be converted to the reactive hydroxyl radical. ROS triggers various signal transduction pathways through activating membrane bound receptors (receptor tyrosine kinases, RTK) for various cytokines (IL-1) and growth factors (EGF, IKGF, TNF-a).which further activate the downstream mitogen-activated protein MAP Kinase signal transduction pathways – a common route shared by many inducers/signals. Receptor activation is mediated in part through ROS-induced inactivation of tyrosine phosphatase whose function is to keep receptors dephosphorylated in an inactive form. For example, under physiological conditions, the EGF receptor exists in an inactive state with low phosphorylation because of its tyrosine phosphatases subunit which removes any phosphates. The hydroxyl radical oxidizes tyrosine phosphatase, rendering it inactive, thus thereby resulting in a constitutively active EGF receptor. The EGF receptor becomes activated within 15 minutes of UV exposure in human skin in vivo and remains activated for at least two hours.

Receptor (i.e. Receptor tyrosine kinases, RTK) activation leads to intracellular signaling (MAP) kinases pathway. There are three classic MAP kinase pathways – ERK (activated by extracellular signal), JNK and p38. UV irradiation activates all three pathways in skin in vivo. The primary end effector of all three MAP kinase pathways is the increased level of transcription factor activator protein-1 (AP-1). The activated AP-1 regulates/activates transcription of several matrix metalloproteinases (MMPs). MMPs are able to degrade many extracellular matrix proteins. Several MMPs are upregulated by AP-1. These include: MMP-1 (collagenase 1), which breaks down types I and III collagen; MMP-9 (gelatinase B), which further breaks down the collagen fragments produced by collagenase; and MMP-3 (stromelysin1), which also degrades collagen. In human skin in vivo, expression of these three MMPs is induced by UV irradiation. This activation of MMPs result in increased collagen breakdown, which has been shown within 24 hours of UV irradiation in vivo. In addition, AP-1 also inhibits collagen synthesis by negatively regulating transcription of type I procollagen genes. mRNA and protein expression of type I procollagen are decreased within eight hours of UV irradiation of human skin in vivo, and become almost absent in the upper dermis within 24 hours of UV irradiation. In addition to receptor activation, ceramide released from the damaged cellular membrane by ROS also activates AP-1 signaling pathway.

UV irradiation enhance collagen degradation and down regulate collagen synthesis not only via the EGF receptor pathway and AP-1 activation. It also block the transforming growth factor-beta (TGF-β) signaling pathway. In skin, TGF-β and TGF-β signaling pathway induces synthesis and secretion of both collagen (collagens I and III) and elastin; and negatively regulates keratinocyte proliferation. TGF-β signaling pathway also downregulate MMPs genes that break down collagen, including MMP-1 and MMP-3. TGF-β exerts its action by binding to its cell surface receptor complex, which is typically composed of TGF-β receptor proteins type I (TβRI) and II (TβRII). When TGF-β binds to TβRII within this complex, it activates the intrinsic serine–threonine kinase activity of TβRI and cascades of signal transduction result in phosphorylation and activation of transcription factors Smad2 and Smad3, which can then combine with Smad4, translocate into the nucleus and regulate/activate genes, including type I collagen. UV irradiation results in downregulation of TβRII gene within eight hours. The decrease or inactivation of TβRII blocks downstream signaling pathways for subsequent activation of type I procollagen. UV irradiation also induces expression of Smad7, which inactivates Smad2 and Smad3. Both TβRII downregulation and Smad7 activation by UV is actually mediated by AP-1; therefore, AP-1 has the direct effect of inducing MMPs, and also has the indirect effect of decrease collagen production through activating Smad7 and/or decreasing TβRII. In addition, inactivation of TGF-β signaling pathway triggers keratinocyte proliferation and epidermal hyperplasia

In addition to activating MAP kinase signaling pathway, receptor tyrosine kinases RTK (activated by ROS as has been mentioned) also activate phosphoinositide3-kinase (PI-3 kinase) signal transduction pathway. Akt/PHEN is the downstream effector of PI-3 kinase pathway where Akt, a kinase is activated and PTEN is inactivated. AKt may activate and regulate AP-1 activity which in turn activate MMPs. PI-3 kinase/Akt pathway has been identified as the effectors of cell survival and proliferation in many cell types. UV induced PI-3/Akt pathway can activate genes in stimulating cellular proliferation.

Photoaged skin is also characterized by an approximately 50% reduction in functional intact collagen while fragmented collagen is increased by about four-fold. The damaged fragmented collagen fiber network further exacerbates the procollagen synthesis by fibroblast. The fragmented collagen surrounding the fibroblast changed mechanical and isometric tension of fibroblast in all directions. fibroblast develops a collapsed and elongated shape. the fibroblast is unable to function appropriately so that procollagen synthesis is decreased. Once initiated, this cycle of degradation of collagen decreased procollagen synthesis is the primary structural basis for the aging skin phenotypes. AP-1 activity is also increased in intrinsic aging process, as are levels of MMP-1 and MMP-9 and levels of degraded collagen. Type I procollagen synthesis is also reduced.

UV irradiation and ROS also activate transcription factor NF-kB. NF-kB are involved mainly in stress-induced, immune, and inflammatory responses and therefore is significant in inflammaging mechanism. UV irradiation and ROS triggered NF-kB activation is also mediated and transduced through MAP kinase pathway (see post “Is NF-kB the Secret to Skin Aging?”). NF-kB signaling activate/regulate proinflammatory cytokines IL-1, IL-6, vascular endothelial growth factor (VEGF) and TNF-b, proteins involved in immunoregulation, and inflammaging mediator COX-2, inducible NO synthase (iNOS). Most proinflammatory cytokines are themselves activators of NF-kB signal transduction pathway upon binding to the corresponding cell surface receptors, thereby forming a positive loop accelerating the activation of genes regulated by NF-kB. NF-kB and/or NF-kB induced proinflammatory molecules stimulates MMPs as well, but primarily is MMP-8. MMP-8 is a collagenase of neutrophil origin, following neutrophil infiltration into UV-irradiated skin. Studies suggest that the main source for UV-induced secretion of MMPs and elastase are infiltrating cutaneous neutrophils.

AP-1 Signaling Activates MMP in Chronological and Photo Skin Aging

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The activator protein 1 (AP-1) is a heterodimeric transcription factor belonging to the c-Fos, c-Jun, ATF and JDP families. It regulates and activates genes in response to a variety of stimuli including stress, pathogen infections, cytokines, growth factors. AP-1 thereby regulates a number of cellular processes including differentiation, proliferation, and apoptosis. AP-1 binds to the TPA DNA response element via its DNA binding domain – leucine zipper DNA binding domain. AP-1 signaling pathway is also involved in the chronological and photo skin aging. The most common stimuli is by the production of reactive oxygen species (ROS). As is known, oxidative stress is one of the main mechanisms for both chronological skin aging and photo skin aging. Increased oxidative stress and accumulation of ROS are observed in intrinsic and extrinsic aging process. ROS not only damage biomacromolecules and cellular components, but also activate the signal transduction pathways which lead to the activation of genes such as the matrix degrading enzymes – matrix metalloproteinase (MMP). Increased level of MMPs are observed in both intrinsic and extrinsic aging process, resulting in the degradation of collagen and elastic fiber networks of the dermal connective tissue.

Dysregulation of intracellular and extracellular homeostasis in the skin by the ROS and the genes activated by ROS induced signal transduction pathway. ROS are necessary participants in multiple MAP kinase pathways. MAP kinase activation results in induction of transcription factor AP-1 that is a major effector of the MAP kinase pathways. AP-1 is increased in aged human skin in vivo and aged skin fibroblasts in vitro. AP-1 transcription factor is composed of two subunits: c-fos and c-jun. In human skin, AP-1 activity is limited by c-jun level while c-fos is constant. c-Jun level is increased in aged skin while c-fos remain same. Among genes regulated by AP-1 are several matrix-metalloproteinase (MMP) and type I procollagen. AP-1 activate MMPs directly and decrease type I procollagen indirectly through activating Smad7. Increased production of MMP-1, MMP-2 (gelatinase A), MMP-3, and MMP-9 occurs in chronologically aged skin and photo aged skin. With the exception of MMP-2, these MMPs are regulated by AP-1. Transcription of several MMP (matrix-metalloproteinase) family members is strongly activated by AP-1. These include MMP-1 (interstitial collagenase or collagenase 1) which initiates degradation of types I and III fibrillar collagens, MMP-9 (gelatinase B), which further degrades collagen fragments generated by collagenases, and MMP-3 (stromelysin 1), which degrades type IV collagen of the basement membrane and activates proMMP-1.

Extracellular Matrix Protein 1 (ECM1) Is Decreased By Chronological Aging and Increased Upon UV Exposure

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Extracellular matrix protein 1 (ECM1) is an extracellular matrix glycoprotein containing motifs with cysteine pattern as the binding sites with other matrix protein or molecules in skin. ECM1 consists of four binding domains for interacting with different ECM molecules ECM1 binds various skin structural and ECM molecules such as perlecan, laminin 332, fibulin-1C/D, fibulin-3 and heparin, as well as dermal interstitial molecules MMP-9, collagen type IV, fibronectin, hyaluronic acid and chondroitin sulphate. ECM1 is one of the proteins connecting the basal layer of epidermis to the basement membrane (BM) and BM to the dermis. ECM1 functions as the “biological glue” in maintaining dermal- epidermal skin structure, skin’s biological function, physiology and homeostasis. Genetic skin diseases suggest a pivotal biological function of ECM1 in human skin – loss of function mutations in the ECM1 gene causes the rare genetic skin disease – the autosomal recessive genodermatosis lipoid proteinosis and the autoimmune disease lichen sclerosus with the presence of ECM1 autoantibody. Similar skin pathologies are observed in both diseases.

ECM1 may be involved in epidermal differentiation because ECM1 gene maps to a region on chromosome where a gene cluster generally known as the “epidermal differentiation complex” is located, containing a remarkable density of genes that fulfill important functions in epidermal differentiation. ECM1a was found in the epidermal basal layer, in dermal blood vessels, the outer root sheath of hair follicles and in sebaceous lobules and epithelium of sweat glands, while ECM1b was expressed in the suprabasal layers of the epidermis. ECM1 binds and interacts with perlecan, an important intrinsic molecule in skin organogenesis. However, in vitro and in vivo experiments revealed no indispensable role for ECM1 in the terminal differentiation process of epidermal keratinocytes. ECM1 is dispensable for terminal differentiation of keratinocytes in unstressed conditions. However, it remains possible that upon stress stimulation, ECM1 may be necessary and essential for the maintenance of epidermal differentiation and homeostasis. Another indication for a possible regulatory role of ECM1b is the presence of two N-glycosylation sites which may bind to epidermal cell surface receptors and may activate certain signal transduction pathways.

ECM1 also plays an important role in the maintenance of the dermal-epidermal communication. ECM1a/c was also found in the network of matrix suprastructures of the basement membrane which is primarily compose of ECM proteins laminin 332 and collagen type IV. The role of ECM1 as a “biological glue” of dermal-epidermal junction structure is base on the fact that ECM1 can bind to most of the important ECM molecules of the basement membrane zone such as perlecan, fibulin-1C-and D, matrix metalloproteinase (MMP)-9 , laminin 332, fibulin-3, collagen type IV, heparin, hyaluronic acid and chondroitin sulphate A. In addition, it also enhances the binding between laminin 332 and collagen IV.

ECM1 is suggested to play a role in skin adhesion, differentiation, scarring, angiogenesis and basement membrane integrity, all of which are important processes in the intrinsic (chronological) as well as the extrinsic (photo) ageing process. ECM1 is one of the many ECM proteins that are significantly decreased in intrinsically aged skin without chronic UV exposure. The decreased ECM1 expression in chronologically aged skin may have profound effects on dermal and epidermal homeostasis, leading to the clinical features of atrophy in both of the skin layers.

Extracellular matrix protein 1 (ECM1) in human skin is decreased by age and increased upon ultraviolet exposure. There are evidence for the involvement of ECM1 in photoaging. A photoprotective role of ECM1 in
skin is proposed based on the research findings. ECM1 was increased in both lower and upper layers of epidermis, induced by chronically UV-exposure, when compared to UV protected skin. UV-irradiation induces fibroblasts to produce more matrix metalloproteinases MMP-1, MMP-3 and MMP-9; MMP-3 degrades many ECM1 binding molecules such as collagen IV, laminin 332, fibronectin and proteoglycans (perlecan). However, the exact mechanism for the proposed photoprotective role of ECM1 is not yet known.

Nrf2 Signaling Coordinates Oxidative Stress Induced Activation of Antioxidant Enzyme Genes

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Oxidative stress plays an important role in the pathophysiology of various age-related diseases and skin aging. Supraphysiological levels of reactive oxygen species (ROS)—such as hydrogen peroxide, superoxide anions, and hydroxyl radicals—severely damage proteins, DNA, and lipids, which results in further tissue damage and organ dysfunction. Environmental stressors (e.g. UV radiation and chronic inflammation) can create and induce excess ROS or free radicals that result in accelerated aging including skin aging. The first line of defense mechanism against excess ROS in the body’s ability to activate and produce natural antioxidant systems within in response to the increased ROS level. There are basically two categories of natural antioxidant system produced in the body for neutralizing ROS: a network of protective enzymatic antioxidants such as glutathione peroxidase, superoxide dismutase, and catalase, and nonenzymatic low-molecular-weight antioxidants such as vitamin E isoforms, vitamin C, glutathione (GSH). Increased amount of ROS upon environmental stimuli will trigger cascade of signal transduction pathways – the result of which is the activation of the genes encoding the antioxidant enzymes. Once the ROS are removed by antioxidant enzymes and ROS level return to physiological level, a negative loop mechanism is activated to turn off the antioxidant enzyme genes.

Nrf-2 signaling pathway is a major mechanism in the cellular defense against oxidative stress. NF-E2-related nuclear factors (Nrf1 and Nrf2) are transcription factors which can bind to a unique DNA sequence in the promoter regions of the antioxidant enzyme genes. This DNA sequence is known as “antioxidant response element”. Transcription of these enzymes is coordinately regulated through antioxidant response elements (AREs). Antioxidant response element (ARE)-mediated induction of antioxidant enzymes is a critical mechanism of protection against chemically induced oxidative stress. Nrf2 is more potent than Nrf1 in activation of ARE-regulated gene activation. Inactive Nrf2 is retained in the cytoplasm by forming the complex to INrf2 (inhibitor of Nrf2) and/or Keap1 . Protein kinase C activation and Map kinase signal transduction routes are involved and activated in Nrf-2 mediated signaling. An increase in oxidative stress leads to the activation of protein kinase C (PKC) and other cytosolic factors. PKC phosphorylation of Nrf2 results in release of Nrf2 from INrf2 and is activated. The activated Nrf2 translocates to the nucleus, forms heterodimers with other proteins, and binds to the ARE. In the nucleus, Nrf2 bind to AREs by interacting with transcription factors in the bZIP family, including CREB, ATF4 and fos or jun. This leads to the coordinated activation of ARE-regulated genes.

Additional nuclear factor including small Mafs (MafG and MafK), large Maf (c-Maf), c-Fos, and Fra1, also bind to ARE and negatively regulate ARE-mediated gene expression. This is the negative loop mechanism for turning off the genes when intracellular ROS is removed and decreased, maintaining a counterbalance to Nrf2 and balancing the oxidation level of the intracellular environment. This is to keep the amount and activities of antioxidant enzymes in check to maintain the cellular defenses active and/or to rapidly restore induced enzymes to normal levels.

There are wide varieties of antioxidant enzymes regulated and activated via Nrf2 signaling. These include, but are not limited to, the following: glutathione transferases, quinone reductase, epoxide hydrolase, heme oxygenase, UDP-glucuronosyl transferases, and gamma-glutamylcysteine synthetase. Oxidative stress is one of the main causes of skin aging as well. There are products on the market that contain ingredients whose activity is targeting the activity of Nrf2. Protandim is one of them which activates Nrf2 and thereby activates the body’s antioxidant enzymes to help protect against oxidative stress. Protandim is clinically proven to reduce oxidative stress in vivo.

The Antioxidant Enzymes Network of The Skin

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The physiological cellular respiration located at mitochondria is always comes with the byproduct of the aerobic metabolism where a small fraction of the oxygen is constantly converted to superoxide anions, hydrogen peroxide, hydroxyl radicals, and other ROS. Although within a certain local concentration range, ROS play important roles in regulating many cellular functions and acting as a secondary messenger to activate specific transcription factors such as NF-κB and AP-1. Aging-associated respiratory function decline can result in enhanced production of ROS in mitochondria. An excess production of ROS, particularly with aging, is harmful to cells that ROS or free radicals can damage proteins, DNAs, cell membranes. To cope with the ROS, body have an internal mechanism of protection and is equipped with a network of protective antioxidants. They include enzymatic antioxidants such as glutathione peroxidase, superoxide dismutase, and catalase, and nonenzymatic low-molecular-weight antioxidants such as vitamin E isoforms, vitamin C, glutathione (GSH).

The antioxidant enzymes network (of the skin) include superoxide dismutase (SOD), catalase (CAT), glutathione system of enzymes, peroxiredoxins, thioredoxin system and methionine reductase. Most antioxidant enzymes are redox enzymes that the active form is an efficient reducing agent for scavenging reactive oxygen species after which the active form become oxidized by ROS and is inactivated. To regenerate the active form of the enzyme, the enzyme system usually has a corresponding reductase (e.g. thioredoxin reductase, glutathione reductase) to regenerate the active enzyme. The activities and concentrations of these antioxidant enzymes and the concentrations of small-molecular-weight antioxidants in blood and tissue cells are altered (mostly declined) in the aging process.

Superoxide dismutases (SODs) are a class of closely related enzymes that catalyze the conversion of the ROS – superoxide anion into oxygen and hydrogen peroxide which is then transformed to water by glutathione peroxidases or by catalase. SOD enzymes are present in almost all cells and in extracellular fluids. Superoxide dismutase enzymes (SOD) require metal ion as cofactors which depend on which isoform it is. Metal ion cofactors can be copper, zinc, manganese or iron. The copper/zinc SOD is present in the cytosol while manganese SOD is present in the mitochondrion. The mitochondrial isozyme seems to be the most biologically important of these three. Copper/zinc SOD (SOD1) may be less important for preserving the longevity.

Catalases are enzymes that catalyze the conversion of hydrogen peroxide (the byproduct of the reaction by SOD) to water and oxygen, using either an iron or manganese cofactor. This protein is localized in peroxisomes in most cells. Catalase is an unusual enzyme since, although hydrogen peroxide is its only substrate, its cofactor is oxidized by one molecule of hydrogen peroxide and then regenerated by transferring the bound oxygen to a second hydrogen peroxide molecule. Despite its apparent importance in hydrogen peroxide removal, humans with genetic deficiency of catalase suffer few ill effects. The natural interaction – synergy – between superoxide dismutases and catalases constitute body’s the most effective system of free radical control .

The glutathione system of antioxidant enzymes include glutathione peroxidases (GPx), glutathione reductase, and glutathione ”S”-transferases. Glutathione peroxidase is another of the body’s major protectors against free radicals – peroxide. The effects of excess cellular peroxidation is diverse and detrimental and must be limited to maintain cellular health. This antioxidant enzyme consists of the amino acid Glutathione and the trace mineral ‘Selenium’. Glutathione peroxidase is an enzyme containing four selenium-cofactors that catalyzes the breakdown of hydrogen peroxide and organic hydroperoxides. Cell membranes consist primarily of lipids which are very susceptible to damage by free radicals, especially peroxide radicals. Lipid peroxides have proven to be toxic. Glutathione peroxidase prevents destruction of cell membranes by removing several classes of lipid peroxides. There are at least four different glutathione peroxidase isozymes. Glutathione peroxidase 1 is the most abundant and is a very efficient scavenger of hydrogen peroxide, while glutathione peroxidase 4 is most active with lipid hydroperoxides. Glutathione ”S”-transferases are particularly abundant in the liver and has strong activity with lipid peroxides removal. Glutathione peroxidases are used primarily to prevent skin related diseases and skin aging. It seems that glutathione peroxidase 1 is dispensable and does not adversely affect life span if it is deficiency.

Peroxiredoxins (Prxs) are peroxidases that catalyze the reduction of hydrogen peroxide, organic hydroperoxides, as well as peroxynitrite. They are divided into three classes: typical 2-cysteine peroxiredoxins; atypical 2-cysteine peroxiredoxins; and 1-cysteine peroxiredoxins. These enzymes share the same basic catalytic mechanism, in which a redox-active cysteine (the peroxidatic cysteine) in the active site is oxidized to a sulfenic acid by the peroxide substrate. Over-oxidation of this cysteine residue in peroxiredoxins inactivates these enzymes, but this can be reversed by the action of sulfiredoxin. Peroxiredoxins seem to be important in antioxidant metabolism and decelerate aging.

The thioredoxin system contains the 12-kDa protein thioredoxin and its companion thioredoxin reductase. The active site of thioredoxin consists of two neighboring cysteines, as part of a highly conserved CXXC motif, that can cycle between an active dithiol form (reduced) and an oxidized disulfide form. In its active state, thioredoxin acts as an efficient reducing agent, scavenging reactive oxygen species (ROS) and maintaining other proteins in their reduced state. After being oxidized, the active thioredoxin is regenerate d by the action of thioredoxin reductase, using NADPH as an electron donor.

Methionine reductase is a unique enzyme that has the ability to remove an extremely toxic free radical known as “Hydroxyl Radical”. The hydroxyl radical is commonly formed through reactions involving heavy metals and other less toxic free radicals, such as mercury reacting with hydrogen peroxide. The hydroxyl radical can damage any type of organic tissue and is considered to be the most dangerous free radical. Hydroxyl radicals are also the main toxins generated by exposure to excessive radiation. With their ability to damage any type of tissue, symptoms directly related to hydroxyl radical induced tissue damage are difficult to identify. It seems that hydroxyl radicals are also formed during exercise in oxygen starved closed rooms or in an auto exhaust filled polluted environments.

Generally, the activities and capacities of antioxidant systems of tissue cells (of the skin) are declined with age, resulting in the accumulation of oxidative damage in the aging process. Although many researchers studied aged-related changes in body’s natural antioxidant defense system, not all results are consistent. The activity of MnSOD located in the mitochondria was found to increase significantly during aging in various tissues. Increased MnSOD resulted in decrease in mitochondrial mass, accumulation of intracellular hydrogen peroxide, and induction of mRNA levels of matrix metalloprotainase-1, – a matrix degrading enzyme that degrade collagen or elastin fibers/extracellular matrix fibers in the skin connective tissue. This suggests that even though superoxide anions produced by mitochondria aerobic metabolism may be scavenged by MnSOD, but hydrogen peroxide thus accumulated in mitochondria may still increase oxidative stress during the aging process. Properly regulated level of of MnSOD is important for cells to cope with oxygen radical-mediated molecular and cellular damage during the aging process. Alterations in the levels of Cu/ZnSOD, CAT, and GPx are also important factors in the aging process. Research indicate that appropriate relative amounts of free radical scavenging enzymes is important for the cellular resistance to oxidative stress. Nutritional programs that contain nutritional factors effective in enhancing the activity of antioxidant enzyme systems have proven to preserve longevity and prevent skin aging.

Telomere Length of the Skin in Association with Chronological Aging and Photoaging

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The cellular telomere length has been the predictor of life span according to the telomere theory of aging. Telomere is the region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration/degradation or from fusion with neighboring chromosomes. Telomeres function by preventing genes located near the the ends of chromosomes from degradation. Some of the telomere is lost – telomere shortening occurs during chromosome replication of each cell division. Over time, due to each cell division, the telomere ends become shorter. When the telomere becomes too short, the chromosome reaches a “critical length” and can no longer replicate at which time enzymes that duplicate DNA cannot continue their duplication due to the lack of binding site. If cells divided without telomeres, genes at the ends of chromosomes can not be copied and replicated. The telomeres are disposable buffers blocking the ends of the chromosomes from degradation and are consumed during cell division. The steady shortening of telomeres with each replication in somatic cells account for the phenomenon of cellular senescence i.e. replicative senescence and hayflick limit – the limitation for somatic cells that can divide before replicative senescence – the fact on which the telomere shortening theory of aging is based. Telomere shortening is body’s natural mechanism to prevent genomic instability and uncontrolled cellular proliferation.

There are mechanisms for the maintenance of telomere length either through activated telomerase – a DNA reverse transcriptase which synthesize telomeres or through ALT (Alternative lengthening of telomeres). The exact mechanism of ALT pathway is not known. Telomerase is an enzyme made of protein and RNA subunits that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes. Telomerase level is very low, almost undetectable activity in most types of somatic cells except the cells that need to divide regularly (e.g., in the immune system). Shorter telomeres and telomerase inactivation has been one of the causes and mechanisms of aging. Many aging-related diseases are linked to shortened telomeres. A variety of premature aging syndromes are associated with short telomeres. Lengthening the telomeres in certain cells through temporary activation of telomerase (by drugs), or possibly permanently by gene therapy can be the approach for developing life extension drugs.

Telomere-based mechanisms of skin aging and photoaging has been elucidated. Intrinsic aging is largely controlled by progressive telomere shortening, compounded by oxidative damage to telomeres and other cellular constituents. Telomere length of the skin is associated with chronological aging, and may associated with photo aging as well. Comparison of telomere lengths from epidermis samples of varying age revealed that telomere length in the epidermis and in the dermis was reduced with age, and average telomere shortening rates in the epidermis and in the dermis are 9 and 11 bp/yr. Increasing evidence indicates that telomerase plays a significant role in maintenance of skin function and proliferation. Telomerase is active in cells of the epidermis but is almost undetectable in the dermis. Mutations in telomerase component genes in the disease dyskeratosis congenita result in numerous epidermal abnormalities. Studies also indicate that telomerase activity in epidermal stem cells might have roles that go beyond telomere elongation.

Accelerated telomere-shortening and telomerase inactivation has been linked with other aging factors and environmental stress. Oxidative stress, which causes DNA damage, has been shown to hasten the shortening of telomeres in cell culture. Evidence indicates that chronic oxidative stress not only causes progressive damage to cellular membranes, proteins, and molecules, but also induces the slowing down of existing telomerase activity and accelerates telomere shortening. The glutathione (GSH)-dependent system is body’s natural antioxidant system to counteract ROS oxidative stress from cellular respiration. Increasing data suggest the role of the GSH-redox antioxidant system in preserving telomeres from oxidative stress damage. Chronic oxidative stress and reduced glutathione, or compromised GSH-redox system is shown to induce a state of progressive telomere shortening. Telomeres in skin cells may be particularly susceptible to accelerated shortening because of both proliferation and DNA-damaging agents such as reactive oxygen species. In sun exposed skin, UV irradiation also damages DNA and accelerates telomere shortening. Aging and photodamage appear to share a common final telomere initiated signal transduction pathway following disruption of the telomere. These telomere-initiated responses, in combination with UV-induced damage to critical regulatory genes, lead to the photoaging phenotypes.

Vitamin D can slow the shortening of the telomeres. Clinical trials found that vitamin D increases telomerase activity. Data suggest that vitamin D may improve telomere maintenance and prevent cell senescence. Telomerase activators are areas of research for developing topical or oral skin care ingredients that are able to stimulate telomerase activity in the skin cells. TA65 – a telomerase activator- is a proprietary extract of Astragalus root. Astragalus root was already used to stimulate the immune system and wound healing. Saponins in the extract are the chemicals responsible for these activities. Further studies found that these saponins stimulate telomerase activity as well. Clinical trials have established life extension effect of TA-65. In addition, in vivo study revealed the consistent improvement in skin assessments in the product group vs placebo group across multiple measures, suggesting a significant effect of TA-65 on skin although skin assessment by vision score were not based on objective measurements of skin structure and function.

Integrin Signaling Mediates Healthy Skin Structure, Physiology And Homeostasis

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Integrins are α β heterodimeric transmembrane receptors that mediate the attachment (adhesion) between a cell and the tissues that surround it, such as other cells or the extracellular matrix (ECM) in the skin epidermis and dermis. There are a number of different α and β subunits identified. Integrins isoforms are named as the combination of their α and β chain numbers. Which ligand in the ECM the integrin can bind to is defined by which α and β subunits the integrin is made of. ECM protein ligands that bind to integrins all contain a RGD (arginine-glycine-aspartic acid) domain which provide the specificity of binding/interaction between ligands and integrin receptors. Common integrin binding proteins in the ECM of the skin include fibronectin, vitronectin, collagen, and laminin.

Integrin signaling pass information about the chemical composition, organization and biomechanical forces of the ECM into the cell and the skin cell responds via a cascade of signal transduction pathways. Therefore, integrins are involved in cell signaling and the regulation of cell cycle, morphology, and motility. Not only do integrins perform this outside-in signaling, but they also operate an inside-out mode in that they reveal the status of the cell to the outside. Integrin not only is available on fibroblast but is also synthesized and located on epidermal keratinocyte. Integrins connect the ECM outside a cell to the cytoskeleton (in particular the microfilaments) inside the cell. Cell attachment to the ECM is a basic requirement to connect ECM and the intracellular actin filamentous system. Focal adhesions (also cell–matrix adhesions or FAs) are specific types of large macromolecular adhesion complexes/assemblies through which both mechanical force and regulatory function of integrin for initiating signaling. Integrin signaling begins with the attachment of ECM to the cell through formation of cell adhesion complexes, which consist of integrins and many cytoplasmic proteins such as talin, vinculin, paxillin, and alpha-actin in. These adhesion complexes attach to the actin cytoskeleton. The integrins thus serve to link two networks across the plasma membrane: the extracellular signals are transmitted. Focal adhesions serve as the mechanical linkages to the ECM, and as a biochemical signaling hub to concentrate and direct numerous signaling proteins at sites of integrin binding and clustering. Next, adhesion complexes relay signal into the cells via their ability to regulate and activate cascades of protein kinases. Integrin signaling pathways can result in and regulate the cell growth, cell division, cell survival, cellular differentiation, and apoptosis.

Integrin-signaling activates pathways on cellular level that are essential in maintaining the functional and healthy skin physiology and homeostasis. An important and still unanswered question is how the surrounding microenvironment in the skin and, in particular, the ECM constituents influence basal keratinocyte and dermis fibroblast behavior during normal homeostasis. Three members of this family are known to be the major receptors for collagens, namely α1β1, α2β1, and α3β1 all of which are synthesized by fibroblasts. They seem to regulate different functions. Integrin α1β1, for example, appears to regulate collagen synthesis, whereas α2β1 mediates collagen gel contraction.

The epidermal cell integrins and their ECM ligands provide a diverse proliferative stimuli for skin basal cells and are key regulators of keratinocyte proliferation and differentiation. Members of basal keratinocytes integrins include: α3β1 (the laminin-5 receptor), α2β1 (the collagen receptor), α5β1 (fibronectin receptor), and αvβ3 and αvβ6 (vitronectin receptors), and α9β1. α9β1 bind to several ligands available beneath migrating keratinocytes only during wound healing. β1 Integrin-mediated adhesion signaling is essential for epidermal progenitor cell expansion. Integrin α6 β4 links to the keratin intermediate filament system in epidermal cells.

During aging, the ECM of the skin dermis (collagen fibers, elastic fibers, ground substance, and cells) and basement membrane (collagen IV, VII, laminin) are changed. Fibers are fragmented or damaged, the fiber organization and alignment are damaged. Adhesion of skin cells to modified ECM structures via specific receptors (integrins) is disturbed; there is no tight connection between cytoskeleton and ECM fibers. Deficiency in integrin-mediated transmembrane signaling and microfilament stress fiber formation by cultured aging dermal fibroblasts were observed. In vivo analysis of fibroblasts sample from different age groups revealed that aged fibroblasts α2β1 integrin (the collagen receptor) amount is not significantly decreased. However, the lines of aged fibroblasts that were poor migrators exhibited a significant reduction in alpha2beta1 activity. Moreover, the lines of aged fibroblasts showed a disordered actin cytoskeleton. Aged fibroblasts with deficient migration are characterized by disorganized cytoskeletal actin and reduced alpha2beta1 function. There is decreased expression of keratinocyte beta1 integrins by epidermal basal cells (basal keratinocyte) in chronically sun-exposed skin in vivo. .

Ingredients that stimulate the integrin signaling has been developed using biomimetic peptide that mimic the ligand conformation containing the integrin binding motif – RGD domain. Cyclopeptide-5 (CP-5) is a biomimetic cyclic oligopeptide that mimic the optimal conformation of the natural ligands of integrin for the stimulation of integrin signaling pathways in skin. The cyclic structure of the peptide provides a rigid, stable and optimal conformation for specificity in binding to integrin. In vitro efficacy studies and DNA microassay (gene chip technology) revealed pathways/process/genes that are activated in the presence of ingredient. Important basement membrane proteins, e.g. laminin IV, collagen IV are increased while ECM enzymatic degradation and MMP levels are decreased.

Base Membrane Laminin And Skin Aging

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The dermal epidermal junction (DEJ) in the skin is the layer responsible for supporting the epidermis and epidermal-dermal adhesion and for the communication between epidermal and dermal cells. Traditionally, the study of the mechanisms of skin aging has overlooked this important area. The DEJ is a basement membrane that separates the skin cells (basal layer of keratinocytes) and anchoring fibril (composed largely of type VII collagen) in/from the epidermis from the extracellular matrix (ECM) in the dermis. This membrane is composed of two layers, the basal lamina, in contact with the epidermal cells, and the reticular lamina in contact with the dermis. The basement membrane contains specific structures/molecules that ensure the stability of the connection and communication between the two major skin compartments. The cohesion between dermis and epidermis is essential to maintain skin balance because it enables the transport of oxygen, nutrients and waste to the epidermis. The basal lamina is abundant in collagen type IV, proteoglycans and the glycoproteins entactin and laminin. These molecules provide a structural network and bioadhesive properties for cell attachment. The membrane, however, serves more than simple structural and filtering roles.

The laminins are a family of multi-domain glycoproteins that are an integral part of the structural scaffolding in almost every tissue of an organism. Laminins are major proteins in the basal lamina of the skin, forming a protein network in the ECM of the base membrane for the maintenance of DEJ architecture and stability. Laminin is, after collagen, the most abundant protein in the ECM of the base membrane at DEJ. The laminins are an important and biologically active part of the basal lamina, influencing cell differentiation, migration, adhesion and cutaneous regeneration. These mechanisms are necessary for skin maintenance and for healing mechanism. Keratinocytes bind to specific domain on Laminin using their own integrin receptors -the transmembrane proteins located on specific junction points called hemidesmosomes. Defects in laminins are involved in some skin pathologies.

Laminins are trimeric proteins that contain an α, β, and γ-chain. The isoforms of laminin molecules vary in chain composition. A number of chain combinations have been identified in vivo. The trimeric proteins can bind to other cell membrane and extracellular matrix molecules as well as bind to other laminins which allows them to form laminin aggregates. Laminins networks are associated with type IV collagen networks via entactin, fibronectin (a glycoprotein), and perlecan (a proteoglycan). They also bind to cell membranes through integrin receptors and other plasma membrane molecules. Through these interactions, laminins critically contribute to cell attachment and differentiation, cell shape and movement, maintenance of tissue phenotype, and promotion of tissue survival. Some of these biological functions of laminin have been associated with specific amino-acid sequences or fragments of laminin.

Studies have confirmed the involvement of the basement membrane in skin aging due to the early alteration of the membrane’s constituents during the aging process. Certain features of the DEJ are altered by the aging process, such as the anchoring ability of keratinocytes with age. Laminin synthesis has also been proved to decrease in aged skin. The production of laminins was reported to be reduced by 31% with age, in
reconstructed skin models. This causes a loss of contact between dermis and epidermis, and results in the skin losing elasticity and becoming saggy. In UV exposed skin, laminins are destroyed, resulting in the disorganization of the DEJ.

Biomimetic peptides are ingredients derived from the skin’s natural protein or peptides that function to mimic the activity of the protein/peptide in vivo. Biomimetic laminin peptides (peptides contain partial laminin sequence) has been developed for topical skin care and cosmetics. In vitro studies concerning the properties of the laminin peptide and its effects on human skin cells has been conducted. Lipotec has developed a laminin peptide ingredient named Serilesine® which is a hexapeptide from the alpha chain of Laminin. This peptide retains many of the characteristics of the native protein, and promotes cell adhesion and proliferation. Serilesine is able to restore the skin’s normal function by promoting synthesis of Laminin, stimulating keratinocyte and fibroblast proliferation, inducing a redensifying effect on the dermis, and an improvement in skin elasticity, compactness, tonicity and smoothness. ChroNOline™ is the commercial anti-aging ingredient manufactured by Atrium which targets specifically to the dermal-epidermal junction functionality. ChroNOline™ is able to boost the production of DEJ components such as collagen VII, laminin, and fibronectin. ChroNOline™ stimulates the production of laminin and fibronectin by human fibroblasts in in vitro and ex vitro studies. Addition of ChroNOline™ to human fibroblasts increased the production of laminins by 26%.

Hyaluronan and Skin Aging

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Hyaluronan (also called hyaluronic acid or hyaluronate or HA) is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. Hyaluronan is a polymer of disaccharides which is composed of D-glucuronic acid and D-N-acetylglucosamine. In the dermal connective tissue, Hyaluronan is the most abundant glycosaminoglycan (GAG) and the main GAG components of the extracellular matrix ground substance – an amorphous viscoelastic gel to hold cells and fibers in place. It is unique among glycosaminoglycan in that it is nonsulfated, and can be very large. Hyaluronan is synthesized by a class of integral membrane proteins called hyaluronan synthases on fibroblast cell surface. There are at least three types of hyaluronic acid synthases: HAS1, HAS2, and HAS3. HAS1 and HAS2 synthases produce high molecular weight HA whereas HAS3 produces low molecular weight HA. Hyaluronic acid is degraded by the enzymes called hyaluronidases. In humans, there are at least seven types of hyaluronidase-like enzymes, several of which are tumor suppressors. The degradation products of hyaluronan (hyaluronan fragments) may have some biological function that is distinct and different from the nonhydrolyzed hyaluronan.

The biological functions of hyaluronan in skin include hydration due to its strong water-binding and retention capacity, cell proliferation and migration, tissue repair, and is also involved in inflammatory or immune response where it can interact and bind to a number of cell surface receptors including its primary receptors CD44 and RHAMM. Hyaluronan also contributes to hydrodynamics and biomechanical properties of the skin. Epidermal hyaluronan also have proinflammatory role and free-radical scavenging function in the inflammatory or immune response which contradicts its role in inflammatory stimulation initially. The amount and synthesis of hyaluronan is significantly increased at the site of tissue injury. Hyaluronan is also abundant in granulation tissue matrix, the perfused, fibrous connective tissue that replaces a fibrin clot in healing wounds. HA plays an important role in the normal epidermis and has crucial functions in the reepithelization processes. Epidermal cells are also able to synthesize HA. It is an integral part of the extracellular matrix of basal keratinocytes, the major constituents of the epidermis that is able to activate keratinocyte proliferation and migration.

The degradation products of hyaluronan, the oligosaccharides and very low-molecular-weight hyaluronan, exhibit pro-angiogenic properties. In addition, recent studies showed hyaluronan fragments (degraded or hydrolyzed form of hyaluronic acid), not the native high-molecular mass of hyaluronan, can induce inflammatory responses in macrophages and dendritic cells in tissue injury. Recent studies have shown that degraded or hydrolyzed form of hyaluronic acid has a strong inhibition of lipid peroxidation and scavenging activities of hydroxyl radical, 1,1-diphenyl-2-picryldydrazyl radical and superoxide anion, suggesting that the reduced size of Hyaluronan has antioxidant property.

The skin content of unfragmented hyaluronic acid decreases with age. The most dramatic histochemical change observed in senescent skin is the evident decrease in epidermal HA. In contract to previous in vitro and in vivo observation, recent studies document that the total HA level remain constant in skin with aging. It is rather the avidity of HA with dermal structure and the HA extractability that changes with aging, suggesting dysfunctional change of HA 3D-molecular structure. The major changes include the increasing avidity and the concomitant loss of extractability. Such intercalated HA may have diminished ability to bind water for hydration. Progressive loss in the size of the HA polymer in skin as a function of age has also been reported. The increased bonding of HA with dermal structure with aging correlates with the progressive crosslinking of collagen and the steady loss of collagen extractability. These changes in structure, size and avidity of hyaluronan result in the clinical signs of aging skin.

When skin is exposed to excessive UVB rays, the cells in the dermis stop producing as much hyaluronan, and increase the rate of its degradation. Hyaluronan degradation products then accumulate in the skin after UV exposure. There is also an abnormal pattern of distribution where HA appears to be deposited on the elastotic material that comprise elastosis and diffusely associated with the actinic damaged collagen.

Topical hyaluronic acid in the form of gels, serums and so forth is widely available and is a very common anti-aging ingredient used in the topical skin care products. Hyaluronic acid-based anti-aging dermal filler are one of the earliest FDA approved wrinkle fillers on the market. Two well known commercially available HA dermal filler is Restylane® (see Restylane Review) and Juvederm® (see Juvederm Review). In addition, hyaluronic acid level may be stimulated by using ingredients that can activate hyaluronic acid synthases or ingredients that can inhibit hyaluronidases (the enzyme degrades HA). However, there is little data and research to prove the availability and effectiveness of such ingredients. One additional approach would be to provide the body with more building blocks for hyaluronic acid, such as glucosamine and N-acetyl-glucosamine. Research is needed, however, to determine whether oral or topical administration of the HA building blocks increases its synthesis in the skin.